Nucleic acids can be divided, according to their strandness, into two major groups comprising single-stranded (ss) or double-stranded (ds) molecules. RNA molecules are most often single-stranded, but the local folding of the polymer chain can result in intra-strand duplexes of different kinds. DNA molecules are usually double-stranded, where the strands are complementary, and form a double helix. Double-stranded nucleic acid molecules are formed by reversible non-covalent interaction between the two strands. The reversibility of complementary binding of nucleic acid strands is crucial for semi-conservative replication of the genetic material and for gene expression.
Analyses of nucleic acids in vitro often rely on their strandness. For example, measurement of renaturation for nucleic acids depends on the ability to monitor the transition from single- to double-stranded form. Further, due to the reversibility of the double-helix, in vitro conditions may facilitate the conversion of double-stranded nucleic acid molecules to single-stranded molecules, or vice versa. Renaturation is an important step in many different methods of molecular biology (e.g. hybridization, PCR and cDNA normalization). It is therefore of great importance to have simple and efficient methods to estimate the strandness of nucleic acid preparations.
A few methods have been described to estimate the amount or to separate/isolate single-stranded and double-stranded nucleic acids from a complex mixture of both. During denaturation or renaturation the transition between single- and double-stranded forms can be monitored by observing changes in UV light absorption due to the hypochromatic effect. The ratio of red to green fluorescence of acridine orange reflects the levels of single- and double-stranded nucleic acids but this ratio also depends on factors such as salt concentration and dye-to-nucleic acid ratio (McMaster and Carmichael 1977; Spano, Bonde et al. 2000). These two methods only allow estimation of the ratio between the single- and double-stranded forms, but they cannot be used for physical separation and isolation of either fraction. They can also not be used to analyse the association between strandness and length of nucleic acid fragments in complex preparations. The strong binding-preference of double-over single-stranded nucleic acids to hydroxyapatite allows the physical separation of single- and double-stranded nucleic acids (Sambrook and Russell 2001). The double-stranded fraction isolated based on the strong hydroxyapatite binding may also contain fragments that are partially single-stranded or completely single-stranded but with local folding resulting in formation of double-stranded structures (e.g. hairpins). Nuclease degradation of single-stranded nucleic acids is often used to discriminate between single- and double-stranded forms in a complex mixture of both. Here only the double-stranded fraction can be recovered and it may contain single-stranded nucleic acids with local double-stranded structures such as stem loops. A major limitation of nuclease degradation is the non-specificity i.e. double-stranded nucleic acids are also nicked and degraded to various extents.
None of the methods described above provide any direct information about the length composition of single-stranded or double-stranded nucleic acid fractions. Further, only the hydroxyapatite method allows isolation of both the single-stranded and double-stranded nucleic acid fractions.
Double-stranded nucleic acid fragments (>50 bp) generally have higher migration velocity than their single-stranded counterparts in polyacrylamide-gel electrophoresis (PAGE). Therefore, double-stranded and single-stranded fragments of equal length will migrate differently and resolve in one-dimensional electrophoresis. This well-known phenomenon has been utilized in e.g. combined heteroduplex/single-stranded-conformation polymorphisms methods (Ravnik-Glavac, Glavac et al. 1994; Sainz, Huynh et al. 1994). All one-dimensional electrophoresis methods based on strandness-dependent separation are limited to samples that contain only a few nucleic acid fragments. If a sample contains many nucleic acid fragments of different lengths, long double-stranded fragments may co-migrate and overlap with shorter single-stranded fragments and thus the population of double-stranded fragments cannot be resolved from the population of single-stranded fragments. This has precluded the use of gel electrophoresis to monitor the strandness of complex nucleic acid preparations.
Methods for separating individual nucleic acid fragments from a complex mixture based on their difference in strandness would be of great interest. Such methods would be much more versatile and powerful if they could be used to simultaneously analyze length distribution of the single- and the double-stranded fractions. Examples where such methods could be used include but are not limited to: I) physical separation of single-stranded and double-stranded nucleic acids fragments allowing, quantification or isolation of either class, II) estimation of the relative amount and length distribution of both single- and double-stranded nucleic acids in biological samples, III) measurement of renaturation kinetics by time-point analysis, IV) isolation of double-stranded nucleic acid fragments containing single-stranded breaks from bulk amount of intact molecules, V) to monitor quality of complex nucleic acid preparations including PCR products and other in vitro amplification products, VI) estimation of cDNA synthesis efficiency and the existence of RNA:DNA hybrids in complex mixtures, and VII) to monitor efficiency of labelling complex nucleic acid samples.
Genetic information is encoded by the linear sequence of bases in a nucleic acid strand. The term “strandness” of nucleic acid molecule is herein used to describe the number of nucleic acid strands are in each nucleic acid molecule. A nucleic acid strand is composed of linear covalently linked poly-nucleotides. Most frequently nucleic acid molecules are single- stranded or double-stranded wherein the double-stranded molecule is formed by reversible intermolecular hydrogen bonding between two single-stranded nucleic acid molecules. In some cases nucleic acid can be multi-stranded e.g. triple helixes or quartets.
As used herein the term “conformation” describes the global 3D structure of nucleic acid molecules. Identical single-stranded nucleic acid molecules can have various different conformations due to e.g. intramolecular hydrogen bonding and folding. Different local intramolecular secondary structures of single-stranded nucleic acids can also affect conformation; hence such differences also fall under the term conformational differences as used herein. Conformational diversity is much more constrained in double-stranded of nucleic acids. Although strandness can affect the overall conformation of nucleic acid molecules, current methods to separate molecules according to conformation cannot by used to separate complex nucleic acid mixtures according to strandness.
The inventors have previously developed a physicochemical method, two-dimensional conformation dependent electrophoresis (2D-CDE) (see, EP 1476549). The method allows separation of double-stranded DNA fragments according to their conformation as well as their length. 2D-CDE is therefore not suitable for separation according to strandness as it is designed for conformational separation of double-stranded nucleic acid molecules. Further conformational differences of double-stranded molecules are ideally enhanced or induced during the first dimension of 2D-CDE while strandness-dependent separation should ideally reduce or eliminate conformational differences within both single- or double-stranded fractions respectively, to ensure separation only according to strandness and length.
Kovar et al. have described a method for “Two dimensional single-strand conformation polymorphism analysis” (Kovar, Jug et al. 1991). The first dimension is carried out under denaturing conditions in order to prevent folding (all double-stranded DNA molecules are made single-stranded). All fragments are therefore single-stranded and migrate strongly according to length as the denaturating condition reduces different conformational variation of each single-stranded nucleic acid molecule. The first dimension is carried out in a capillary electrophoresis system. After the first dimension the capillary gel matrix is laid onto a non-denaturating polyacrylamide gel matrix in a horizontal gel electrophoresis system. During the second dimension electrophoresis all nucleic acid molecules are single-stranded as in the first dimension. Due to lack of denaturating agents in the second dimension the single-stranded molecules can adapt various conformations and the separation will by according to both fold-back conformation and length. The method can however not be used to separate single- and double-stranded linear nucleic acid molecules. The method only allows separation according to different length and fold-back conformation of single-stranded fragments.